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Departments of
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Neurobiology and
Neurology, University of California School of Medicine, Los Angeles, CA 90095
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A protective effect of testosterone is thought to underlie why males are less susceptible to autoimmune disease than females. This is based on studies that include removing testosterone from male mice via castration as well as by treatment of female mice with testosterone. For example, castration of male nonobese diabetic mice resulted in an increased prevalence of diabetes (15), and castration of male SJL mice increased EAE severity (16). Conversely, female nonobese diabetic or SJL mice implanted with testosterone pellets had a lower incidence of diabetes and less severe EAE, respectively, compared with those implanted with placebo pellets (17, 18). Also, castration of males increased the incidence and severity of thyroiditis and adjuvant arthritis, whereas testosterone treatment was protective (8, 9). Furthermore, testosterone treatment of female MRL/Mp-lpr/lpr mice with Sjogren’s syndrome reduced lymphocyte infiltration into lacrimal tissue (10). Finally, in systemic lupus erythematosus in NZB/NZW mice, testosterone treatment improved survival (13).
Previous studies have indicated that gender differences in
susceptibility may be due to gender differences in cytokine production
upon autoantigen-specific stimulation. In males, compared with females,
greater Th2 and less Th1 cytokine production has been observed
(11, 18, 19). The balance between cytokines produced by
Th1 and Th2 lymphocytes is considered central to the development of
autoimmune disease. Th1 lymphocytes produce IFN-
, IL-2,
lymphotoxin-
, and TNF-. Th2 lymphocytes secrete IL-4, IL-5, IL-6,
IL-10, and IL-13. These two cell types are mutually inhibitory, and
their development occurs under very specific conditions. If a naive T
lymphocyte is initially stimulated with Ag in the presence of IL-12,
the immune response is skewed toward Th1. However, if a naive T
lymphocyte is initially stimulated with Ag in the presence of IL-4, the
immune response is skewed toward Th2. In this manuscript we address the
mechanisms responsible for gender differences in the Th1/Th2
balance.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female and male SJL mice, aged 4 wk, were obtained from The
Jackson Laboratory (Bar Harbor, ME). Female C57BL/6 and IL-10-,
IFN--, and IL-12b-deficient mice on the C57BL/6 background, aged 4
wk, were also obtained from The Jackson Laboratory.
Hormone pellets
Ninety-day release pellets of 5-dihydrotestosterone (DHT) at
doses of 5 and 15 mg, as well as placebo pellets that contain the
carrier binder (cholesterol-methyl cellulose--lactulose) were
purchased from Innovative Research of America (Sarasota, FL). Pellets
were implanted s.c. in the scapular area of the neck of gonadally
intact female mice at 4 wk of age using a trochar after methoxyflurane
inhalation anesthesia. Mice were implanted with pellets for 2 wk before
splenocyte isolation.
Serum testosterone levels
Two weeks after implantation, blood was obtained by intracardiac puncture from placebo- or DHT-treated mice. Serum testosterone levels of individual mice were determined by ELISA in duplicate according to the manufacturer’s directions (Oxford Biomedical Research, Oxford, MI).
Cytokine profiles
Splenocytes were cultured in 24-well plates (Costar 3524;
Corning Glass, Corning, NY) at 5 x 106
cells/ml in 2 ml RPMI 1640, without phenol red (BioWhittaker,
Walkersville, MD), supplemented with Nutridoma-NS (Roche, Indianapolis,
IN), HEPES, L-glutamine, nonessential amino acids, sodium
pyruvate, and antibiotics (all obtained from BioWhittaker). Cells were
stimulated with Ab to CD3 (anti-CD3, 0.2 or 1 µg/ml) either
alone or in conjunction with Ab to CD28 (anti-CD28) (2.5 µg/ml;
anti-CD3 (clone 145-2C11) and anti-CD28 (clone 37.51), both obtained
from BD PharMingen, San Diego, CA) or with medium alone. Culture
supernatants were harvested at 24 and 48 h, and ELISAs were
performed for various cytokines. IFN-, IL-4, IL-10, and IL-12 p40
were quantified using a sandwich ELISA technique. The Ab pairs were
DB-1 and a rabbit anti-mouse polyclonal (IFN-; BioSource
International, Camarillo, CA), 11B11 and BVD6-24G2 (IL-4, BD
PharMingen), JES5-2A5 and JES5-16E3 (IL-10; BD PharMingen), and C17.8
and C15.6 (IL-12 p40; BD PharMingen). The sensitivity ranges for the
ELISA were 8–500 pg/ml for IFN-, 15–2000 pg/ml for IL-4, 15–2000
pg/ml for IL-10, and 30–4000 pg/ml for IL-12.
Enrichment for splenocyte cell subpopulations
Splenocytes were isolated from female SJL mice and stimulated with 1 µg/ml anti-CD3. After 24 h in culture, nonadherent cells were harvested to obtain T lymphocytes and B lymphocytes for further purification into subpopulations. Adherent cells were used as the macrophage-enriched subpopulation and were 70% MAC-1 positive cells as determined by flow cytometry. Nonadherent cells were purified further into CD4+ T lymphocyte and CD8+ T lymphocyte and B lymphocyte subpopulations by MACS as described previously (20, 21). Briefly, cells were resuspended in MACS buffer (PBS containing 2 mM EDTA, 0.5% BSA, and 0.01% sodium azide) and separated into four groups. The first group of cells was not manipulated further and constituted the unseparated cell population. It consisted of 31–47% B lymphocytes, 31–39% CD4+ T lymphocytes, and 6–19% CD8+ T lymphocytes. The second, third, and fourth groups of cells were incubated for 15 min at 4°C with magnetic bead-conjugated Abs specific for B220 to label B lymphocytes, CD4 to label CD4+ T lymphocytes, or CD8 to label CD8+ T lymphocytes (Miltenyi Biotec, Auburn, CA). After two washes in MACS buffer, cells were added to a positive selection column (VS+, Miltenyi Biotec). The column was washed four times with buffer. Cells retained in the column were then eluted, and flow cytometric analysis showed that cells incubated with B220 Ab were 94–96% B lymphocytes, cells incubated with CD4 Ab were 96–99% CD4+ T lymphocytes, and cells incubated with CD8 Ab were 93–99% CD8+ T lymphocytes.
Cell depletions
Spleens were isolated from female SJL mice that had been implanted with placebo or DHT pellets 2 wk before the experiment and dissociated into a single-cell suspension in MACS buffer. These cells were incubated for 15 min at 4°C with magnetic bead-conjugated Abs specific for CD4. After two washes in MACS buffer, cells were added to a depletion column (BS; Miltenyi Biotec). The cells that flowed through contained only 1–4% CD4+ T lymphocytes as assessed by flow cytometry and were used as the CD4+ T lymphocyte-depleted fraction. The cells that were retained in the columns were further purified on a positive selection column (RS+, Miltenyi Biotec) and were 90–94% CD4+ T lymphocytes. These cells were used as the CD4+ T lymphocyte fraction.
RT-PCR for IL-10 and androgen receptor (AR) within each enriched cell subpopulation
Total RNA from each subpopulation that had been enriched for a certain cell type or depleted of a certain cell type was prepared by guanidinium isothiocyanate extraction followed by phenol-chloroform extraction. One microgram of total RNA was reverse transcribed using the GeneAmp RNA PCR kit from PerkinElmer (Branchburg, NJ). cDNA was amplified for 30 cycles for IL-10 and actin or for 35 cycles for AR, using primers for murine IL-10 (CLONTECH, Palo Alto, CA), murine AR (sequence obtained from Ref. 19 , primers synthesized by AnaGen Technologies, Palo Alto, CA), or for murine actin CLONTECH). Southern blots of the PCR products were hybridized using a 32P-labeled murine IL-10 internal probe (CLONTECH) or a 32P-labeled murine AR internal probe (5'-TAC CAG CTC ACC AAG CTC CT-3', synthesized by AnaGen Technologies). The radiolabeled Southern blot was exposed to a phosphor storage screen (Molecular Dynamics, Sunnyvale, CA) and read using the PhosphorImager (Molecular Dynamics) as previously described (22).
In vitro treatments with DHT
In the primary stimulation, splenocytes from male SJL mice were
placed in culture in the presence of 10-9 M DHT
or the hormone diluent alone, and the cells were stimulated with
anti-CD3 (1 µg/ml) and anti-CD28 (2.5 µg/ml) for 72 h. At that
time, cells were harvested, pelleted, and replaced in culture in the
presence of 25 U/ml IL-2. After 5 days cells were again harvested. A
majority of these cells were pelleted and replaced in culture, either
alone or in the presence of irradiated splenocytes. The remaining cells
were analyzed by flow cytometry to determine the percentage of viable
cells (10%), and the percentage of viable cells that were
CD4+ T lymphocytes (80%). During this secondary
stimulation, the medium contained either 10-9 M
DHT or hormone diluent alone as a control, and the cells were again
stimulated with anti-CD3 (1 µg/ml) and anti-CD28 (2.5 µg/ml).
Culture supernatants were harvested at 24 h, and ELISAs were
performed for IL-10 and IFN-.
Statistical analysis
Mean cytokine levels and serum testosterone levels were calculated from duplicate measurements and compared using paired t tests, one-way ANOVA, repeated measures ANOVA, and 2 x 2 factorial ANOVA.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our laboratory has previously demonstrated that male SJL mice
develop less severe adoptive EAE (12), and that
splenocytes from male mice in the effector phase of the disease secrete
more IL-10 when stimulated with myelin basic protein (MBP) than
splenocytes from female mice (18). Our initial experiments
were designed to determine whether this difference in IL-10 secretion
was specific to T lymphocytes stimulated with MBP or was a more
generalized phenomenon. Naive splenocytes from healthy male and female
mice were stimulated with anti-CD3, and cytokine secretion was measured
by ELISA. Fig. 1
shows concentrations of
IL-4, IL-10, IL-12, and IFN- in supernatants from such cultures of
three male-female pairs. The Th2 cytokine IL-4 was secreted at close to
2-fold higher levels by splenocytes from male mice, which was a
statistically significant difference (p =
0.04). The Th2-associated cytokine IL-10 was also secreted at close to
2-fold higher levels by splenocytes from male mice, also reaching
statistical significance (p = 0.02). In
contrast, the Th1-inducing cytokine IL-12 was secreted at significantly
lower levels by anti-CD3-stimulated splenocytes isolated from males
compared with females (p = 0.04). Secretion of
the Th1 cytokine IFN- was not significantly different. Secretion of
IL-5 and IL-6 was also not significantly different (data not
shown).
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The possibility that testosterone was the factor responsible for
the difference in cytokine secretion between splenocytes from each
gender was tested by implanting normal female mice with 90-day release
DHT pellets 2 wk before an experiment. DHT was chosen instead of
testosterone because testosterone can be converted to estrogen, whereas
DHT cannot. Therefore, the effect of DHT is purely androgenic. In
initial experiments mice were treated with either of two doses of DHT
(5- or 15-mg pellets) to determine which would increase the serum
testosterone level in female mice to that in male mice. As Fig. 2 shows, implanting female mice with
either the 5- or the 15-mg pellet caused an increase in the serum
testosterone level compared with that in female mice implanted with a
placebo pellet. Most importantly, there was no significant difference
between the serum testosterone level in female mice treated with 5 mg
DHT and that in untreated male mice, whereas the serum testosterone
level in the female mice treated with 15 mg DHT was supraphysiologic.
Because of these findings, the physiologic DHT dose of 5 mg was used in
subsequent experiments.
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B indicates, splenocytes from
female mice treated with DHT secreted significantly more IL-10 than
splenocytes from placebo-treated female mice (p
< 0.0001). Splenocytes from female mice treated with DHT also secreted
significantly less IL-12 (p = 0.03; Fig. 3C). Therefore, treatment of female mice with DHT
recapitulated the increase in IL-10 and the decrease in IL-12 secretion
seen in Fig. 1
in male mice compared with female mice. These data
indicate that differences in the levels of testosterone in male mice vs
female mice are probably responsible for the difference in IL-10 and
IL-12 secretion between genders. In contrast, treatment of female mice
with DHT did not recapitulate the difference in IL-4 secretion seen
between genders, indicating that increased testosterone levels in males
were not responsible for the increased IL-4 secretion (Fig. 3A). As in Fig. 1D, treatment of female mice with
DHT did not alter IFN- production (Fig. 3D).
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To address the mechanism by which testosterone altered cytokine production, the first step was to determine how DHT caused both an increase in IL-10 and a decrease in IL-12 secretion. It was possible that DHT was acting independently to increase IL-10 secretion and decrease IL-12 secretion. However, because IL-10 and IL-12 can regulate each other (23, 24), it was also possible that DHT was acting to increase IL-10 secretion, with increased IL-10 levels causing a decrease in IL-12 secretion. Conversely, DHT may have initially decreased IL-12 secretion, with decreased IL-12 levels then causing an increase in IL-10 secretion.
To investigate whether DHT was altering IL-10 secretion independently
of changes in IL-12 secretion, female IL-12 knockout mice
(IL-12-/-) and female C57BL/6 wild-type mice
were implanted with DHT or placebo pellets. After 2 wk,
their splenocytes were isolated and stimulated in culture with
anti-CD3. Splenocytes from IL-12-/- female mice
treated with DHT secreted significantly more IL-10 than splenocytes
from placebo-treated IL-12-/- female mice
(p = 0.03; Fig. 4A). This indicated that DHT
increased IL-10 secretion in a manner that was not dependent upon
differences in IL-12.
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B). In addition, there was no
gender difference in IL-10 and IL-12 production upon stimulation with
anti-CD3 of C57BL/6 female mice vs male mice (data not shown). There
was also no difference in IL-12 production upon stimulation of
splenocytes from DHT vs placebo-treated
IL-10-/- mice and no difference in IL-10 and
IL-12 production upon stimulation of splenocytes from DHT vs
placebo-treated IFN--/- C57BL/6 mice (data
not shown). However, conclusions concerning the role of IL-10 and
IFN- in DHT-induced differences in IL-12 production cannot be drawn
from these data because the absence of a difference in cytokine
production between DHT and placebo treatment in the
IL-10-/- and IFN--/-
mice may merely reflect the absence of a difference in cytokine
production inherent to the C57BL/6 background.
In contrast, although the absence of differences in cytokine production
upon DHT treatment of the IL-10-/- and
IFN--/- mice is not informative, the
presence of cytokine differences upon DHT treatment of
IL-12-/- mice is informative. Indeed, the
observation that IL-10 production is increased upon DHT treatment of
IL-12-/- mice confirms that the increase in
IL-10 during DHT treatment is not dependent upon DHT-induced
differences in IL-12 production. Furthermore, these data provide new
insights. Removing IL-12, as occurs in the IL-12 knockout, unmasked the
ability of DHT to increase IL-10 in the C57BL/6 strain. In contrast,
removing IFN-, as occurs in the IFN- knockout, did not unmask
this ability of DHT to increase IL-10 in this strain. This suggests
that IL-12 regulation of IL-10 may override the ability of testosterone
to increase IL-10 in some strains (C57BL/6), but not in others (SJL).
Further studies in our laboratory will pursue the role of IL-12 in
suppressing the effect of testosterone in some strains but not
others.
IL-10 is expressed by CD4+ T lymphocytes
Our data indicated that DHT was affecting IL-10 secretion by
splenocytes, independently of changes in IL-12 secretion. T
lymphocytes, B lymphocytes, and macrophages all have the potential to
produce IL-10. Thus, in further delineating the mechanism of action of
testosterone, it became important to determine which cell type within
splenocyte cultures was expressing IL-10. Following 24 h of
stimulation with anti-CD3, splenocytes from female placebo- and
DHT-treated mice were enriched, using MACS, into populations of
CD4+ T lymphocytes, CD8+ T
lymphocytes, and B lymphocytes. Adherent cells were used as a
macrophage-enriched fraction. Each enriched cell subpopulation was then
assessed for IL-10 production by RT-PCR. As demonstrated in Fig. 5, RT-PCR analysis for IL-10 within each
enriched cell population demonstrated the greatest IL-10 signal in the
CD4+ T lymphocyte population. Furthermore,
when the splenocyte population was depleted of
CD4+ T lymphocytes, the majority of the IL-10
signal was eliminated. The low level signal in the
CD8+ T lymphocyte, B lymphocyte, and macrophage
fractions could be due to either low level expression of IL-10 by these
cell populations or low level contamination of these fractions with
CD4+ T cells. Identical results were obtained
from DHT-treated mice (data not shown). Together these data strongly
suggest that IL-10 is predominantly expressed by
CD4+ T cells.
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To determine whether IL-10 was also predominantly expressed by
CD4+ T lymphocytes at the protein level, cell
populations obtained from either placebo-treated female mice or
DHT-treated female mice were enriched for CD4+ T
lymphocytes or were depleted of CD4+ T
lymphocytes using MACS. The four populations of cells were placed in
culture and stimulated for 48 h with anti-CD3 and anti-CD28.
ELISAs were then performed to determine the IL-10 levels in the culture
supernatants. Significantly more IL-10 was secreted from cultures of
cells enriched for CD4+ T lymphocytes than from
cultures of cells depleted of CD4+ T lymphocytes
(Fig. 6). This was true for both cells
obtained from mice treated with placebo pellets as well as cells
obtained from mice treated with DHT pellets. Consistent with previous
results, cultures of cells enriched for CD4+ T
lymphocytes produced significantly more IL-10 if they were isolated
from mice treated with DHT pellets as opposed to mice treated with
placebo pellets. Conversely, the lack of a difference in IL-10
secretion between the CD4+ T lymphocyte-depleted
population treated with DHT vs placebo indicated a lack of a DHT
treatment effect on other cell types within the spleen. Together these
results further support the conclusion that it is
CD4+ T lymphocytes that are producing IL-10, and
that it is the CD4+ T lymphocytes that are
reacting either directly or indirectly to a change in testosterone
levels in the mice.
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For testosterone to have a direct effect on
CD4+ T lymphocytes, these cells would need to
express the receptor for testosterone, the AR. Therefore, AR expression
was analyzed by RT-PCR on RNA obtained from enriched splenocyte
populations. The AR was expressed by enriched populations of
CD4+ T lymphocytes, CD8+ T
lymphocytes, and macrophages (Fig. 7),
consistent with previous reports (19, 25). Enriched
populations of B lymphocytes expressed only low levels of AR. Because
it is likely that AR expression at the mRNA level indicates AR
expression at the protein level, these data give further support to the
hypothesis that testosterone could be acting directly on
CD4+ T lymphocytes via the AR to increase IL-10
expression.
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To fully test the hypothesis that testosterone is acting directly
on CD4+ T lymphocytes, we treated naive
CD4+ T lymphocytes in vitro with DHT. Splenocytes
were stimulated in the presence of a physiologic dose of DHT in both a
primary and a secondary stimulation. Flow cytometry verified that cells
used in the secondary stimulation were comprised predominantly of
CD4+ T lymphocytes. As shown in Fig. 8A, addition of DHT to these
cultures of CD4+ T lymphocytes caused a
significant increase in IL-10 secretion. IFN- secretion was not
significantly changed (data not shown). This first set of data was
obtained from cells cultured on irradiated splenocytes, raising the
question as to which cells were responding to DHT with an increase in
IL-10: the CD4+ T lymphocytes or the irradiated
splenocytes. Therefore, these experiments were repeated in the absence
of irradiated splenocytes (Fig. 8B). Again, cultured
CD4+ T lymphocytes, in the absence of irradiated
splenocytes, produced significantly more IL-10 when DHT was added in
vitro. Therefore, it is clear that DHT can act directly on
CD4+ T lymphocytes to cause an increase in IL-10
secretion.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-12, supporting the conclusion that the male
immune system is shifted toward Th2 immunity (7, 18, 19, 26). Mechanisms underlying why there is a gender difference in
cytokine production remain unknown. Many possibilities exist. They
include differences in levels of male sex hormones such as
testosterone, differences in female sex hormones such as estrogen and
progesterone, and differences in genes located on sex chromosomes.
To delineate such mechanisms, we believed it was necessary to use a
simple system. Indeed, although cytokine studies of gender differences
in EAE collectively showed increased Th2 cytokines and decreased Th1
cytokines in male cells compared with female cells, studies differed
slightly with regard to which cytokines were altered. This was most
likely due to differences in experimental methods: the use of peptide
vs whole molecule as stimulatory autoantigens, the use of proteolipid
protein vs MBP, active vs adoptive EAE, and the presence or absence of
adjuvant, which induces artificial Th1 conditions. Comparing cytokine
production between splenocytes derived from normal male and female mice
after stimulation with anti-CD3 eliminated all these complicating
factors. Thus, we first wanted to determine whether there were gender
differences in cytokine production in this simpler system. Similar to
studies in EAE, we found that levels of the Th2 cytokines IL-4 and
IL-10 were higher and the IL-12 level was lower in splenocytes from
males compared with females. However, in this study there was no
difference in IFN- production.
Having established that there were gender differences in our system, we next wanted to determine which cytokine differences were due to testosterone. Splenocytes from female mice implanted with testosterone pellets, like splenocytes from male mice, secreted more IL-10 and less IL-12. However, treatment with testosterone did not cause increased IL-4 production. This clearly indicates that testosterone does not recapitulate all the cytokine differences seen in male vs female mice, and that the increase in IL-4 must be due to gender differences in other sex hormones and/or genes found on sex chromosomes.
Regarding the relevance of these cytokine changes to EAE pathogenesis,
IL-12 can mediate EAE through an IFN--independent mechanism
(24, 27, 28), whereas the role of IFN- in EAE is
controversial (29, 30, 31, 32, 33, 34). Thus, a decrease in IL-12
production associated with testosterone treatment may indeed be
important in the decreased susceptibility of male mice to EAE. The
finding of increased IL-10 production is equally as important as the
finding of decreased IL-12 production upon testosterone treatment.
Numerous studies have shown that IL-10 is essential in the regulation
of EAE. Specifically, treatment of EAE with IL-10 has been shown to
ameliorate disease (35, 36, 37), whereas administration of Abs
to IL-10 has exacerbated disease (35, 38). Although
treatment of EAE with IL-4 also ameliorated disease (39, 40), studies of IL-4- and IL-10-deficient mice and IL-4 and
IL-10 transgenic mice have shown that IL-10 may play a more critical
role in protection from EAE. Indeed, IL-10-/-
mice developed more severe EAE compared with wild-type mice, and
overexpression of IL-10 rendered mice resistant to EAE
(41). In contrast, although some studies have shown
increased susceptibility of IL-4-/- mice to
EAE, others have not (41, 42, 43), and transgenic mice
overexpressing IL-4 had disease similar to that in wild-type mice
(41). Because IL-10 has been shown to play a protective
role and IL-12 a disease-promoting role in EAE, and because
testosterone increases IL-10 and decreases IL-12, testosterone would
appear to play an important role in gender differences in
susceptibility to this disease.
Having shown that testosterone increases IL-10 and decreases IL-12 production, it is important to determine the mechanism through which this occurs. There are three central questions that need to be answered: 1) is the decrease in IL-12 secretion upon treatment with testosterone responsible for the increase in IL-10 secretion? 2) what cell type within spleen has altered cytokine production? and 3) are the effects of testosterone on this cell direct or indirect? In answer to the first question, our data from IL-12-/- mice showed that the increase in IL-10 secretion is independent of changes in IL-12 secretion. Furthermore, it is possible that in the SJL strain, the decrease in IL-12 production was secondary to the increase in IL-10 production. This hypothesis is supported by a recent report in which treatment of male SJL mice with Abs to IL-10 resulted in increased IL-12 production (44). Taken together, these studies indicate that testosterone initially increases IL-10 production, which may then lead to a decrease in IL-12 production.
To address the second question of what cell type within spleen has altered cytokine production, we have presented evidence in this manuscript at both the RNA and protein levels indicating that CD4+ T lymphocytes are the predominant cell type within spleen that produce and secrete IL-10. Furthermore, it is the IL-10 produced by the CD4+ T lymphocytes that is responsive to alterations in testosterone levels both in vivo and in vitro.
Finally, the third question, is the effect of testosterone direct or
indirect? To this end, it is essential to determine which cells within
the splenocyte population express the AR. RT-PCR analysis showed that
CD4+ T lymphocytes express the AR, supporting the
possibility of direct action of testosterone on these cells. However,
the AR is also expressed by CD8+ T lymphocytes
and macrophages. Thus, an indirect action of testosterone mediated
through these cells was also possible. To definitely demonstrate that
testosterone can act directly on CD4+ T
lymphocytes, we showed that in vitro stimulation of
CD4+ T lymphocytes in the presence of
testosterone and in the absence of other cells resulted in increased
IL-10 production. This is consistent with previous studies that have
shown that treatment of naive T lymphocytes from V
8.2 TCR transgenic
mice with testosterone in vitro makes more IL-10 upon stimulation with
MBP Ac1–11 peptide (19). Taken together, these studies
show that testosterone can act directly on CD4+ T
lymphocytes to increase IL-10 production.
In summary, although many cells within spleen express the AR, we have
shown that testosterone can act directly upon
CD4+ T lymphocytes to increase IL-10 expression
during stimulation with anti-CD3. The increase in IL-10 from
CD4+ T lymphocytes may then lead to reduced IL-12
production by macrophages. We have no evidence to suggest that
testosterone can act directly on macrophages to cause a decrease in
IL-12. However, the mechanism presented here does not rule out the
possibility that testosterone may also affect
CD8+ T lymphocytes or macrophages. Indeed, a
recent report using in vitro cultures of T lymphocytes and peritoneal
exudate cells at a ratio of 9:1 has suggested that this can occur
following stimulation with 10- to 50-fold higher concentrations of
anti-CD3 (10 µg/ml) (44). Notably, however, we found
that testosterone treatment had no effect on IL-10 production by
CD8+ T lymphocytes and macrophages cultured ex
vivo (Fig. 6). Furthermore, the increase in IL-10 during in vitro
treatment of CD4+ T lymphocytes with testosterone
occurred in the absence of these other cells and thus was not dependent
upon interactions with them.
The differences in cytokine production between males and females
(increased IL-4 and IL-10, decreased IL-12) could be hypothesized to be
due to a testosterone-induced shift in the immune system toward Th2
immunity. Furthermore, it could be hypothesized that to cause a general
shift, testosterone may be affecting one or more of the key molecules
involved in establishing cell lineage, such as GATA-3 or STAT6.
However, this is not the case. Although the expression of many
cytokines is different between genders, testosterone initially affects
only IL-10. Thus, testosterone is not acting on molecules involved in
establishing cell lineage; rather, it is acting by altering regulation
of the IL-10 gene. In support of this idea, a search for an androgen
response element in the promoter for the IL-10 gene revealed a putative
response element in the human IL-10 gene promoter, 
300 bp upstream
of the TATA box. However, this putative response element in not well
conserved in the mouse IL-10 gene promoter, with only 8 of the 12
nucleotides being identical with the consensus sequence. Although other
nonconserved sequences in various genes have been shown to be androgen
responsive (45, 46, 47), it is difficult to speculate about
whether the putative androgen response element in the IL-10 promoter is
indeed androgen responsive until promoter analysis experiments are
conducted. Nonetheless, the observations presented in this manuscript
regarding the effect of testosterone on up-regulation of IL-10 in
CD4+ T lymphocytes are informative in and of
themselves and are likely to be relevant to a variety of autoimmune
disease models characterized by gender differences.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Rhonda R. Voskuhl, Department of Neurology, University of California Multiple Sclerosis Center, 750 Westwood Plaza, Los Angeles, CA 90095. E-mail address: rvoskuhl{at}ucla.edu
3 Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; DHT, 5-dihydrotestosterone; AR, androgen receptor; MBP, myelin basic protein.
Received for publication September 22, 2000. Accepted for publication June 6, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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M. C. Siracusa, M. G. Overstreet, F. Housseau, A. L. Scott, and S. L. Klein 17{beta}-Estradiol Alters the Activity of Conventional and IFN-Producing Killer Dendritic Cells J. Immunol., February 1, 2008; 180(3): 1423 - 1431. [Abstract] [Full Text] [PDF]
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 N. L. Sicotte, B. S. Giesser, V. Tandon, R. Klutch, B. Steiner, A. E. Drain, D. W. Shattuck, L. Hull, H.-J. Wang, R. M. Elashoff, et al. Testosterone Treatment in Multiple Sclerosis: A Pilot Study Arch Neurol, May 1, 2007; 64(5): 683 - 688. [Abstract] [Full Text] [PDF]
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 S. E. Dunn, S. S. Ousman, R. A. Sobel, L. Zuniga, S. E. Baranzini, S. Youssef, A. Crowell, J. Loh, J. Oksenberg, and L. Steinman Peroxisome proliferator-activated receptor (PPAR){alpha} expression in T cells mediates gender differences in development of T cell-mediated autoimmunity J. Exp. Med., February 19, 2007; 204(2): 321 - 330. [Abstract] [Full Text] [PDF]
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 J J Corrales, M Almeida, R Burgo, M T Mories, J M Miralles, and A Orfao Androgen-replacement therapy depresses the ex vivo production of inflammatory cytokines by circulating antigen-presenting cells in aging type-2 diabetic men with partial androgen deficiency. J. Endocrinol., June 1, 2006; 189(3): 595 - 604. [Abstract] [Full Text] [PDF]
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 J. Caron, L. Lariviere, M. Nacache, M. Tam, M. M. Stevenson, C. McKerly, P. Gros, and D. Malo Influence of Slc11a1 on the Outcome of Salmonella enterica Serovar Enteritidis Infection in Mice Is Associated with Th Polarization. Infect. Immun., May 1, 2006; 74(5): 2787 - 2802. [Abstract] [Full Text] [PDF]
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J. Reddy, H. Waldner, X. Zhang, Z. Illes, K. W. Wucherpfennig, R. A. Sobel, and V. K. Kuchroo Cutting Edge: CD4+CD25+ Regulatory T Cells Contribute to Gender Differences in Susceptibility to Experimental Autoimmune Encephalomyelitis J. Immunol., November 1, 2005; 175(9): 5591 - 5595. [Abstract] [Full Text] [PDF]
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 K. M. Palaszynski, D. L. Smith, S. Kamrava, P. S. Burgoyne, A. P. Arnold, and R. R. Voskuhl A Yin-Yang Effect between Sex Chromosome Complement and Sex Hormones on the Immune Response Endocrinology, August 1, 2005; 146(8): 3280 - 3285. [Abstract] [Full Text] [PDF]
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A. C. Roden, M. T. Moser, S. D. Tri, M. Mercader, S. M. Kuntz, H. Dong, A. A. Hurwitz, D. J. McKean, E. Celis, B. C. Leibovich, et al. Augmentation of T Cell Levels and Responses Induced by Androgen Deprivation J. Immunol., November 15, 2004; 173(10): 6098 - 6108. [Abstract] [Full Text] [PDF]
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 C. J. Malkin, P. J. Pugh, R. D. Jones, D. Kapoor, K. S. Channer, and T. H. Jones The Effect of Testosterone Replacement on Endogenous Inflammatory Cytokines and Lipid Profiles in Hypogonadal Men J. Clin. Endocrinol. Metab., July 1, 2004; 89(7): 3313 - 3318. [Abstract] [Full Text] [PDF]
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M. Gylling, E. Kaariainen, R. Vaisanen, L. Kerosuo, M.-L. Solin, L. Halme, S. Saari, M. Halonen, O. Kampe, J. Perheentupa, et al. The Hypoparathyroidism of Autoimmune Polyendocrinopathy-Candidiasis-Ectodermal Dystrophy Protective Effect of Male Sex J. Clin. Endocrinol. Metab., October 1, 2003; 88(10): 4602 - 4608. [Abstract] [Full Text] [PDF]
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). RNA was isolated from each cell subpopulation, and 1 µg RNA
was reverse transcribed with primer sets specific for IL-10. Data shown
are from a Southern blot of the RT-PCR products hybridized with an
IL-10 internal probe and are representative of experiments repeated
twice.
